Phosphorylation of GluR1, a subunit of AMPA receptors, is an important mechanism for regulating the strength of synaptic transmission in the brain. Two sites on the C-terminus of GluR1 are phosphorylated, potentiating AMPA receptor currents through different mechanisms. Ser831 is phosphorylated by CaM-KII and increases the single channel conductance. Ser831 phosphorylation is increased during LTP and may contribute to synaptic potentiation in the hippocampus. Ser845 is phosphorylated by PKA and increases the open-probability of GluR1. Ser845 phosphorylation does not change during LTP, but is decreased during LTD, suggesting that the two phosphorylation sites are distinctly regulated during bi-directional synaptic plasticity. However, some past studies and the preliminary results in this study indicate that S845 is a poor substrate for PKA in vitro. Thus, the main goal of this project is to determine the absolute phosphorylation state of GluR1 at Ser831 and Ser845 in cultured hippocampal neurons under a variety of conditions and to correlate these phosphorylation states with functional regulation of AMPA receptor properties (single-channel conductance, open probability, number of receptors).